The present invention relates generally to separation of rare cell populations from whole blood samples. More particularly, the present invention provides a system and non-invasive method for enriching the populations of rare cells, such as nucleated fetal erythrocytes or nucleated fetal red blood cells ("NRBCs") obtained from maternal blood samples by separating the NRBCs from the mother's erythrocytes, leukocytes and other blood components. Those skilled in the art will appreciate that the present system and method are not confined, in their utility, to enrichment of fetal NRBCs. Rather, a wide variety of rare cell populations present in the peripheral blood circulation may be separated according to the present method, as will be explained more fully herein.
In accordance with a preferred embodiment thereof, the present invention offers a system and method for enriching a population of fetal-origin NRBCs from a maternal blood sample by a negative selection process which concentrates the NRBCs by lysis and yields viable rare cell populations which may be cultured or otherwise analyzed. While enrichment of viable fetal-origin NRBCs is described herein as an operative model of the present system and method, the present invention is not so limited in its utility.
Physicians have long sought to develop non-invasive methods for prenatal diagnosis because the available methods, amniocentesis and chorionic villus sampling (CVS) are potentially harmful to the mother and to the fetus. The rate of miscarriage for pregnant women undergoing amniocentesis is increased by 0.5-1%, and that figure is be slightly higher for CVS. Because of the inherent risks posed by amniocentesis and CVS, these procedures are offered primarily to older women, i.e., those over 35 years of age, who have a statistically greater probability of bearing children with congenital defects.
Some non-invasive methods have already been developed to diagnose specific congenital defects. For example, maternal serum alpha-fetoprotein, and levels of unconjugated estriol and human chorionic gonadotropin can be used to identify a proportion of fetuses with Downs syndrome, however, it is not one hundred percent accurate. Similarly, ultrasonography is used to determine congenital defects involving neural tube defects and limb abnormalities, but is useful only after fifteen weeks gestation.
The presence of fetal cells in the maternal peripheral circulation has been known for some time. However, fetal cells represent a rare cell population in maternal circulation present in estimated numbers ranging from 1 in 100,000 to 1 in 1,000,000. Fetal lymphocytes (Walknowska, et al., Lancet 1:1119 (1969), Schroder, et al., Blood 39:153 (1972; Schroder, Scand J. Immunol. 4:279 (1975)), syncytiotrophoblasts (Douglas et al., Am. J. Obstet. Gynecol. 78:960 (1959); Goodfells, et al., Brit. J. Obstet. Gynecol. 89:65 (1982); Covone, et al., Lancet 2:841 (1984); Kozma, et al., Hum. Reprod. 1:335 (1986)), cytotrophoblasts (Mueller, et al., Lancet 336:197 (1990)), and erythrocytes (Freese, et al., Obstet. Gynecol., 22:527 (1963); Clayton, et al., Obstet. Gynecol. 23:915 (1964); Schroder, J. Med. Genet. 12:230 (1975; Medaris, et al., Am. J. Obstet. Gynecol. 148:290 (1984)) have been identified in the maternal peripheral blood circulation.
Separation of nucleated fetal erythrocytes from maternal blood has been identified as a desirable method for facilitating prenatal diagnosis of genetic disorders. Fetal NRBCs have been separated from maternal blood by flow cytometry using a lysing reagent (European Published Patent Application No. 582736, published Feb. 16, 1994); by triple gradient discontinuous gradient gel electrophoresis (Bhat, et al, U.S. Pat. No. 5,275,933, issued Jan. 4, 1994); by separation from nucleated cells using leukocyte depletion and ammonium chloride lysis of enucleated erythrocytes (Goldbard, PCT Publication WO 9417209, published Aug. 4, 1994); by use of anti-CD71 monoclonal antibody and magnetic beads and in-situ fluorescence hybridization (FISH) (Ahlert, et al, German Published Patent Application No. 4222573, published Aug. 12, 1993) or by other antibodies specific to a fetal erythrocyte antigen (Bianchi, PCT Publication WO 9107660, published May 30, 1991). Unfortunately, to date, there are no clinically acceptable methods for prenatal genetic diagnosis using maternal peripheral blood samples. Amniocentesis and CVS continue to be the only options available to a small percentage of pregnant women. These women represent the group statistically at risk of conceiving a fetus having genetic abnormalities.
Substantial attention has been given to methods of prenatal sex diagnosis using fetal cells in the maternal peripheral blood. Because obtaining a maternal blood sample is far less invasive than amniocentesis, CVS or fetal blood sampling, methods which are capable of enriching fetal cells from maternal peripheral blood samples have become a paramount focus of neonatology. Many studies have been made on prenatal sex diagnosis by means of the polymerase chain reaction (PCR) amplifying the Y-chromosome-specific DNA sequence in maternal blood. See, e.g., Bianchi, D. W., et al., Proc. Natl. Acad. Sci. USA, 87:3279-3283 (1990), Bianchi, D. W., et al. Prenat. Diagn., 13:293-300 (1993), Wachtel, S., et al., Hum. Reprod., 6:1466-1469 (1991), Suzemori, K., et al., Obstet. Gynecol., 80:150-154 (1992). In some of the studies using PCR amplification, fetal cells in the maternal blood were enriched and analyzed by PCR. While the sensitivities and specificities of the methods reported in the PCR studies is high, e.g., 64-100 and 80-100 percent, respectively, and the positive and negative predictive values were 75-100 and 67-100 percent, respectively, the diagnostic accuracy reported in each study was insufficient to be acceptable for routine clinical use. Hamada, H., et al., Prenat. Diag., 15:78-81 (1995).
Flow cytometry has also been used to separate fetal NRBCs on the basis of positive CD71 antibody (transferrin receptor) and glycophorin-A antibody binding. PCR conducted on the flow-sorted cells identified male fetuses at a rate of 100% accuracy and female fetuses at a rate of 83% accuracy. Simpson, J. L., et al., J. Am. Med. Ass'n. 270:2357-2361 (1993). Immunogenetic procedures have been combined with fluorescence-activated cell sorting (FACS) procedures to enrich fetal cells in whole blood from pregnant women. Herzenberg, L. A., et al., Proc. Natl. Acad. Sci. USA, 76:1453-1455 (1979) employed rabbit anti-HLA-A2 antibodies in conjunction with fluorescein-conjugated goat anti-rabbit immunoglobulins and analyzed the fluorescein-bound cells using FACS.
Fluorescence in situ hybridization (FISH) offers a method for identifying desired DNA in a cell at the interphase stage. FISH with conventional cytogenetic methods has been used on fetal cells recovered from maternal blood samples for sex determination, Wessman, M., et al., Prenat. Diag., 12:993-1000 (1992), and to detect chromosomal abnormalities, Simpson, J. L., et al., Prenat. Diag., 12:S12 (Supp. 1992) fetal trisomy 21!, Bianchi, D, et al, Prenatal Diagnosis through the Analysis of Fetal Cells in the Maternal Circulation, Genetic Disorders and the Fetus 3d Ed., Milunsky, A., ed., pp. 759-770 (1992) Fetal trisomy 18!.
Fetal progenitor cells have also been enriched from maternal blood by ligand binding onto an immobilization medium. CellPro, Inc., International Publication No. WO 94/25873, published Nov. 10, 1994, discloses an immunoselection method for enriching fetal erythroid progenitor cells from maternal blood by separating a large fraction of maternal erythrocytes from the blood sample, such as by density gradient centrifugation on a Ficoll gradient or by preferential lysis of maternal erythrocytes in the presence of ammonium chloride, potassium chloride and a carbonic anhydrase inhibitor, then incubating a sample of maternal blood with a ligand to bind fetal progenitor cells and then removing unbound blood products, leaving the enriched, ligand-bound fetal erythroid progenitor cells. The ligands disclosed include antibodies, erythropoietin or transferrin. The ligand is immobilized on any of a variety of solid supports, such as hollow fibers, magnetic beads, plates, dishes, flasks, meshes, screens, solid fibers, membranes or dipsticks.
The CellPro International Publication, discloses, for example, the use of first member-second member binding pairs, including biotin-avidin, biotin-streptavidin, biocytin-avidin, biocytin-streptavidin, methotrexate-dihydrofolate reductase, 5-fluorouracil-thymidylate synthetase and riboflavin-riboflavin binding protein, wherein the first binding member is linked to a ligand capable of binding fetal nucleated erythroid cells, the second binding member is linked to an immobilization medium, and the second member has a binding affinity constant for the second member of greater than about 10.sup.8 M.sup.-. The preferred embodiments disclosed include methods for positive and negative selection of fetal cells, useful independently or in conjunction with one another. The positive selection process using a binding pair consisting of biotinylated anti-CD34 antibody and immobilized avidin. The CD34 antigen is expressed on fetal progenitor cells and hematopoietic progenitor cells, however, in normal adults, the hematopoietic progenitor cells reside in the bone marrow and CD34 positive cells are found in the peripheral circulation at a rate of less than 0.1%. The negative selection process using a binding pair consisting of biotinylated anti-CD45 and immobilized avidin. The CD45 antigen is expressed on maternal erythrocytes, but not fetal progenitor cells. The immunoselected fetal cells then may be subjected to analysis by karyotyping, PCR, RFLP, SSCP or FISH to provide genetic or biochemical information. The CellPro method, however, suffers from a clinically unacceptable yield of enriched cells. While this published application states enrichment of fetal progenitor cells to a concentration greater than 1%, the highest level of enrichment supported by the examples was attained by a dual positive selection process using sequential CD34 antibody binding steps yielding a sample containing about 1 in 2,000 or 0.5% fetal cells, representing about a 500-fold enrichment from the starting sample.
To date, however, no clinically acceptable method for enrichment of rare cell populations, particularly fetal nucleated erythrocytes, from peripheral blood samples has been devised which yields cell populations sufficient to permit clinical diagnosis. The clinical need for a method capable of producing higher yields of rare fetal cell populations separated from maternal whole blood was recently underscored by Hamada, H., et al., Prenat. Diag. 15:78-81 (1995) who stated that "The data obtained in this study suggest that fetal sex determination using maternal peripheral blood with FISH is possible and that this diagnostic method will be clinically useful when more cells are analyzed."